Agents that act to damage DNA by alkylation, DNA-DNA crosslinking, DNA-protein crosslinking, and/or DNA cleavage have been used as potent chemotherapeutic agents. Some of these agents are reductively activated by electron transfer to a moiety such as semi-quinone. Agents which are thought to be reductively activated include the naturally occurring and synthetic mitomycins and the enediynes such as neocarzinostatin. Kasai et al., SynLett, 10:788 (1992) (mitomycins); and Nicolaou et al., Angewandte Chemie, 30:1387 (1991) (enediynes). Cellular resistance to these types of chemotherapeutic agents develop, especially in tumor cells. The mechanism of resistance of tumor cells and the organisms that produce the agents are not known.
Mitomycins are agents that are reductively activated and catalyze DNA alkylation and DNA-DNA crosslinks. Mitomycins are antitumor antibiotics produced by Streptomyces lavendulae and other Streptomyces species. Several mitomycins have been characterized including the naturally occurring mitomycin A, mitomycin B, mitomycin C (MMC), porfiromycin, and mitiromycin. The structural properties and biological activity of mitomycins have motivated a large number of studies to determine cellular target sites and mechanism of action. Complete structural characterization of MMC has been followed by studies showing the basis of its remarkable activity against mammalian tumor cells. Iyer et al., Science, 145:55 (1964); Schwartz et al., Science, 142:1181 (1963). The molecule has three important functional groups, which include a quinone ring system, carbamate moiety, and a highly strained aziridine group that contribute together to determine MMC target site specificity and ability to alkylate DNA. Significantly, the precise regions in DNA that undergo mono- and bifunctional alkylation by MMC, leading to inhibition of replication and subsequent cell death have also been determined. Cera et al., Biochemistry, 28:3908 (1989); Kumar et al., Biochemistry, 32:1364 (1993); Teng et al., Biochemistry, 28:3901 (1989); and Tomasz et al., Science, 235:1204 (1987).
Mitomycins are DNA bioreductive alkylating agents which present a difficult and unique challenge for cellular resistance in producing microorganisms. Beijnen et al., J. Pharm. Biochem. Anal., 4:275 (1986); Kumar et al., supra. Evidence strongly supports the idea that the first step in biological activation of mitomycins occurs by catalytic electron transfer to the benza-quinone species to form a semi-quinone radical shared by mitomycins. Hoey et al., Biochemistry, 27:2608 (1988). In this form, the molecule would fit into the minor groove of DNA and undergo further reduction to the hydroquinone. Beijnen et al., supra.; Hoey et al., supra. The second electron transfer is presumed to occur with rapid kinetics, and is followed by DNA alkylation through activation of the two electrophilic centers in the mitomycin hydroquinone species. With this unique biological activity, it is clear that the two most common resistance mechanisms in bacteria, protection of the target site (methylation of ribosomal RNA) and modification of the antibiotic (phosphorylation or acetylation), are unlikely to be applicable to mitomycin. Davies, FEMS Microbiology Reviews, 39:363 (1986); Witt et al., Appl. Microbiol., 13:361 (1990). Specifically, it would be impractical for the cellular DNA to be protected (e.g. modified by extensive methylation) because the GC content in S. lavendulae is xcx9c70%, and the mitomycin target site for mitomycin C (MMC) has been shown specifically to be CpG residues. Teng et al., supra. Likewise, protection by drug modification through phosphorylation or acetylation would not prevent electron transfer or the activation of electrophilic centers in the molecule.
Although there is a wealth of knowledge about the structure, mode of action and mechanism of activation of mitomycin C, no studies have been conducted to determine the molecular basis for resistance in the producing organisms such as S. lavendulae. Indeed, resistance mechanisms are unknown for the entire class of bioreductive alkylating and cleaving agents like MMC, and other potent anti-tumor compounds that alkylate or cleave DNA following reductive activation. Beijnen et al., supra.; Hoey et al., supra.; and Woo et al., J. Amer. Chem. Soc., 115:1199 (1993). Understanding resistance to these molecules in these producing microorganisms may provide insight into the problem of multidrug resistance (MDR) of cancer cells, and its effect on long term therapeutic efficacy of antineoplastic agents. Moscow et al., Multidrug Resistance. Cancer Chemotherapy and Biological Response Modifiers Annual 11, Elsevier Science Publishers B.V. (1990). Identification of additional mechanisms that contribute to broad spectrum drug resistance of tumors in mammalian systems may allow the development of strategies to identify and effectively control this complex problem.
Thus, there is a need to study cellular resistance to compounds that bioreductively alkylate or cleave DNA. There is a need to identify agents that inhibit resistance to compounds that bioreductively alkylate or cleave DNA. There is also a need to identify and modify DNA gene sequences that are responsible for drug resistance mechanisms in microorganisms and in animal and human tumor cells.
The invention provides for an expression cassette and vectors including the expression cassette. The expression cassette comprises a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent operably linked to a promoter functional in the cell. The preferred DNA bioreductive alkylating or cleaving agent is a mitomycin. Preferred DNA sequences are those that substantially correspond to the mcr and mrd loci of S. lavendulae B619. The promoter is preferably functional in Streptomyces and provides for a sufficient level of gene expression so that resistance of the cell to the DNA bioreductive alkylating or cleaving agent can be detected.
Once a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent is identified, it can be used to generate DNA probes. A DNA probe has sufficient complementarity to all or a portion of a known DNA or RNA sequence that provides resistance to the agent so that it can hybridize to the DNA or RNA sequence, preferably under low stringency conditions. Portions of a DNA or RNA sequence preferably are restriction endonuclease fragments of the mcr or mrd DNA sequence. The preferred probes are complementary to the 6.7 kb BclI fragment of pDHS3000 encoding mcr and the 4.2 kb BclI fragment of pDHS3001 encoding mrd.
Two loci have been identified that provide mitomycin resistance to mitomycin sensitive host cells. One locus found on a 6.2 kb BclI fragment from S. lavendulae has now been designated mcr and is the same as the locus designated mcrA in U.S. application Ser. No. 08/133,963. On the mcr locus, three open reading frames were identified and are now designated 1) mcrA which is the same as the DNA sequence identified as mcrA1; 2) mcrB which is the same as the DNA sequence identified as mcrA2; and 3) mcrORF3 which is the same as the DNA sequence previously identified as mcrAORF3 in U.S. application Ser. No. 08/133,963. The other locus is found on a 4.2 kb BclI fragment on plasmid pDHS3001 and is now referred to as mrd and is the same as the locus previously identified as mcrB in U.S. application Ser. No. 08/133,963. The identifiers of the gene loci and DNA sequences in this application have been changed from the parent application Ser. No. 08/133,963 as described above. Subject matter from the parent application that referred to the previous identifiers has been modified to the new identifiers, but the gene loci and DNA sequences remain the same as those disclosed in the parent application Ser. No. 08/133,963.
The invention also provides for polypeptides and antibodies specific for the polypeptides. A polypeptide can be encoded by a DNA sequence that provides resistance to a cell to a DNA bioreductive alkylating or cleaving agent such as the mcr DNA sequence. The preferred polypeptide is the MCRA polypeptide which is about a 56,000 dalton polypeptide encoded by mcrA.
The invention also provides transformed cells. Transformed cells comprise an expression cassette comprising a DNA sequence that substantially corresponds to a DNA sequence that provides resistance to the cell to a DNA bioreductive alkylating or cleaving agent operably linked to a promoter. The preferred cell is a cell sensitive to the DNA bioreductive alkylating or cleaving agent such as Streptomyces lividans. The preferred DNA sequence substantially corresponds to the DNA sequence of the mcrA and mcrB genes. The expression cassette preferably is expressed in an amount sufficient to confer resistance to the cell to the agent.
The invention also provides methods for identifying agents that inhibit the resistance of the cell to the DNA bioreductive alkylating or cleaving agent. One method involves using transformed cells. Transformed cells resistant to the agent comprise an expression cassette as described herein. The transformed cells are incubated with an effective amount of an agent suspected to inhibit resistance of the cell to the DNA bioreductive alkylating or cleaving agent and an effective amount of the DNA bioreductive alkylating or cleaving agent. After incubation for a suitable amount of time, it can be determined if the suspected agent inhibited the resistance of the cell to the DNA bioreductive alkylating or cleaving agent.
In an alternative version, an inhibitory agent can be identified by its ability to inhibit the function of a polypeptide encoded by the DNA sequence. A substantially pure polypeptide such as MCRA is incubated with a DNA sample and the DNA bioreductive alkylating or cleaving agent and the inhibitory agent. After incubation, it can be determined whether the inhibitory agent inhibited the function of MCRA polypeptide by measuring the binding of the DNA bioreductive alkylating or cleaving agent to the DNA sample or by determining whether DNA alkylation has occurred. If the suspected inhibitory agent inhibits the function of MCRA, binding and/or activity of the DNA bioreductive alkylating or cleaving agent is increased in the presence of the suspected inhibitory agent, preferably 2 to 20-fold.
The invention also provides a method for identifying sequences homologous to the mcr or mrd sequences in other cell types and/or organisms. Preferably, the cells are multi-drug resistant or mitomycin C-resistant tumor cells. The method involves generating a DNA library and amplifying selected sequences in the library using polymerase chain reaction. Amplification of selected sequences is accomplished by selecting oligonucleotide primers that are complementary to a portion of the mcr or mrd loci. Once formed, the amplified products are isolated and screened for homology to mcr or mrd by hybridization to a DNA probe. Sequences that hybridize can be mapped using restriction enzymes and sequenced using standard methods.